2007 Annual Report
1a.Objectives (from AD-416)
The objectives of this proposed research will be to identify naturally occurring genetic variation in Salmonella enterica that correlates with the evolution of egg contamination and enhanced growth of the bacterium in the hen, the egg and in the on-farm environment and to then determine how different genetically defined strains vary in their pathobiology within the hen and how these differences affect the risk of egg contamination and the control of disease.
1b.Approach (from AD-416)
Our approach will be to identify single nucleotide polymorphisms (SNPs) that occur naturally in the genome of Salmonella enterica, link them to the phenotypic attributes of the pathogen that are relevant to causation of egg contamination and growth to high cell density, and develop a phylogenetic database that aids cost effective screening of the Salmonellae for these traits. We will then characterize the processes by which bacteria are deposited inside eggs laid by infected laying hens and assess the significance of these processes for proposed cost effective and feasible disease control measures such as egg refrigeration, diagnostic egg culturing, and assay of shell quality.
Confirmation of small scale polymorphisms within the genome of Salmonella enterica serovar Enteritidis (S. Enteritidis). 23 new polymorphisms were reported in Version 4 of the NCBI database on July 31, 2007. To date, a total of 217 single nucleotide polymorphisms (SNPs) and other small polymorphisms have been confirmed by forward/reverse sequencing of 3 key strains as differentiating phenotypes of S. Enteritidis. There are another 230 putative polymorphisms remaining to be confirmed.
Egg-contaminating S. Enteritidis is resistant to beta-lactam antibiotics. A panel of 240 antibiotics at 4 different concentrations (960 conditions) was analyzed for the ability to inhibit growth of different phenotypes of S. Enteritidis. This work was done in collaboration with the BEAR unit.
Assignment of PFGE patterns to genome and phenotype. Another collaboration with the BEAR unit revealed that the 3 genomes of S. Enteritidis that have been analyzed by mutational mapping, resequencing and confirmatory sequencing were representative of the 1st, 4th and 10th most prevalent isolates in the United States as determined by pulsed field gel electrophoresis (PFGE).
Determination of limits of detection by Nimblegen resequencing technology. Three isolates of S. Enteritidis PT13 obtained from different hosts (human, plant, chicken) were analyzed by mutational mapping. Fifty of the sites reported as significantly different were sequenced, but no differences were found. These results suggest that DNA of bacteria either generates local perturbations to primer hybridization or that epigenetic modifications introduce variation.
In vitro penetration of egg yolks by Salmonella strains during 36-hour ambient temperature storage. In a laboratory egg contamination model, S. enteritidis and S. heidelberg strains penetrated through the egg yolk membrane and most strains grew to significantly higher levels inside the egg yolk contents during incubation at 30 C, but significantly less penetration and growth were observed at 20 C.
Colonization of specific regions of the reproductive tract and deposition at different locations inside eggs laid by hens infected with Salmonella. In experimentally infected laying hens, the frequency of S. enteritidis and S. heidelberg colonization greater higher in the upper region of the reproductive tract (the ovary) than in lower regions of this tract (the oviduct), but the frequency of Salmonella isolation from eggs laid by these hens was not significantly different in the yolk or albumen.
Completed work associated with NFCA Number 58-6612-7-160 (6612-32000-04 00N). This project is a high-throughput analysis of the ability of 940 mutant strains of S. Typhimurium to successfully colonize the internal organs and intestinal contents of chickens. Only samples that yielded an initial cell concentration of > 4000 post-infection of birds were adequate for downstream processing. USDA has completed its share of the work associated with this project. MONITORING ACTIVITIES: Communications were maintained by email exchange and sharing of results by spreadsheet.
Subpopulations of S. Enteritidis which vary in the ability to contaminate eggs have different antibiotic resistance profiles. Simple user-friendly methods for differentiation of S. Enteritidis subpopulations are needed to enable other scientists, especially those involved in epidemiological analyses, to observe complex interactions between strains that have highly similar genomes but different phenotypes. The antibiotic resistance profiles of two S. Enteritidis subpopulations and a reference S. Enteritidis PT4 strain were compared to strains of S. Typhimurium, S. Heidelberg and S. Newport using conventional resistance assays and phenotype microarrays. The antibiotics tetracycline and ampicillin were useful for differentiating subpopulations of S. Enteritidis that vary in the ability to contaminate eggs and to grow to high cell density. Results strongly suggest that antibiotic resistances of S. Enteritidis emerge from small scale genetic polymorphisms that escape detection by fingerprinting methods. This research addresses National Program 108 (Food Safety), Component 1.1 (Pathogens, Toxins, and Chemical Contaminants - Preharvest), Problem Statement 1.1.1 (Methodology).
In vitro penetration of egg yolks by Salmonella strains during 36-hour ambient temperature storage. We determined that S. enteritidis and S. heidelberg strains were able to penetrate through the egg yolk membrane and most strains grew to significantly higher levels inside the egg yolk contents during incubation at 30 C, but significantly less penetration and growth were observed at 20 C. Although chickens infected with Salmonella do not deposit this pathogen inside egg yolks very often, bacteria from the surrounding albumen might penetrate through the membrane that surrounds the yolk, resulting in rapid and extensive Salmonella growth in the nutrient-rich interior contents of the yolk prior to egg refrigeration. We used a laboratory egg contamination model to assess the ability of S. enteritidis or S. heidelberg strains to penetrate through the yolk membrane and multiply inside yolks during incubation at warm temperatures to simulate the conditions of a proposed national S. enteritidis control program that would allow unrefrigerated storage of eggs on farms for up to 36 hours. These results demonstrate that controlling ambient temperatures during pre-refrigeration storage may be an important adjunct to prompt refrigeration for limiting Salmonella growth in eggs and thereby for preventing egg-transmitted human illness. This research addresses National Program 108 (Food Safety), Component 1.1 (Pathogens, Toxins, and Chemical Contaminants - Preharvest), Problem Statement 1.1.3 (Ecology, Host-Pathogen, and Chemical Contaminants Relationships).
Colonization of specific regions of the reproductive tract and deposition at different locations inside eggs laid by hens infected with Salmonella. We determined that the frequency of S. enteritidis and S. heidelberg colonization in infected hens was higher in the upper region of the reproductive tract (the ovary) than in lower regions of this tract (the oviduct), but the frequency of Salmonella isolation from eggs laid by these hens was not significantly different in the yolk or albumen. Salmonella deposition inside eggs appears to result from the infection of reproductive organs (the ovary and oviduct) of laying hens, but little is know about how infection of specific regions of the reproductive tract affects the location of the resulting contamination inside eggs. We experimentally infected groups of laying hens with large oral doses of S. heidelberg or S. enteritidis strains and subsequently determined the frequencies at which these organisms colonized different regions of the reproductive tract and were deposited in different locations inside eggs. These results demonstrate that no distinctive patterns of deposition in eggs can be identified that are attributable to any corresponding patterns of infection of particular parts of the reproductive tract by individual Salmonella strains. This research addresses National Program 108 (Food Safety), Component 1.1 (Pathogens, Toxins, and Chemical Contaminants - Preharvest), Problem Statement 1.1.3 (Ecology, Host-Pathogen, and Chemical Contaminants Relationships).
5.Significant Activities that Support Special Target Populations
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Morales, C., Gast, R.K., Bouldin, J.G. 2006. J. Linkage of avian and reproductive tract tropism with sequence divergence adjacent to the 5s ribosomal subunit rrfh of salmonella enterica.Federation of European Microbiological Societies Microbiology Letters.264(1):48-58.
Gast, R.K., Guraya, R., Bouldin, J.G., Holt, P.S. 2007. In Vitro Penetration of Egg Yolks by Salmonella Enteritidis and Salmonella Heidelberg Strains During 36-hour Ambient Temperature Storage. Poultry Science.86(7):1431-1435
Gast, R.K., Guraya, R., Bouldin, J.G., Holt, P.S., Moore, R.W. 2007.Colonization of specific regions of the reproductive tract and deposition at different locations inside eggs laid by hens infected with salmonella enteritidis or salmonella heidelberg. Avian Diseases.51(1):40-44.
Bouldin, J.G., Musgrove, M.T., Humphrey, T.J., Cates, C.W., Gast, R.K., Morales, C. 2007. Pathotyping of salmonella enterica by analysis of snps in cyaa and flanking 23s ribosomal sequences. Environmental Microbiology.9(4):1047-1059.
Guard, J.Y., Morales, C. 2006. Evolutionary trends in two strains of Salmonella enterica subsp. I serovar Enteritidis PT13a that vary in virulence potential. National Center for Biotechnology Information (NCBI). Available at:http://www.ncbi.nlm.nih.gov/genomes/static/Salmonella_SNPS.html.
Guard, J.G., Morales, C. 2007. Evolutionary trends in two strains of Salmonella enterica subsp. I serovar Enteritidis PT13a that vary in virulence potential. National Center for Biotechnology Information (NCBI). Available:http://www.ncbi.nlm.nih.gov/genomes/static/Salmonella_SNPS.html